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Abstract

Background

Chronic Fatigue Syndrome (CFS) is a severe, systemic illness characterized by persistent,
debilitating and medically unexplained fatigue. The etiology and pathophysiology of
CFS remains obscure, and diagnosis is formulated through the patient’s history and
exclusion of other medical causes. Thereby, the availability of biomarkers for CFS
could be useful for clinical research. In the present study, we used a proteomic approach
to evaluate the global changes in the salivary profile in a couple of monozygotic
twins who were discordant for CFS. The aim was to evaluate differences of salivary
protein expression in the CFS patient in respect to his healthy twin.

Methods

Saliva samples were submitted to two-dimensional electrophoresis (2DE). The gels were
stained with Sypro, and a comparison between CFS subject and the healthy one was performed
by the software Progenesis Same Spot including the Analysis of variance (ANOVA test).
The proteins spot found with a ≥2-fold spot quantity change and p<0.05 were identified
by Nano-liquid chromatography electrospray ionization tandem mass spectrometry. To
validate the expression changes found with 2DE of 5 proteins (14-3-3 protein zeta/delta,
cyclophilin A, Cystatin-C, Protein S100-A7, and zinc-alpha-2-glycoprotein), we used
the western blot analysis. Moreover, proteins differentially expressed were functionally
analyzed using the Ingenuity Pathways Analysis software with the aim to determine
the predominant canonical pathways and the interaction network involved.

Results

The analysis of the protein profiles allowed us to find 13 proteins with a different
expression in CFS in respect to control. Nine spots were up-regulated in CFS and 4
down-regulated. These proteins belong to different functional classes, such as inflammatory
response, immune system and metabolism. In particular, as shown by the pathway analysis,
the network built with our proteins highlights the involvement of inflammatory response
in CFS pathogenesis.

Conclusions

This study shows the presence of differentially expressed proteins in the saliva of
the couple of monozygotic twins discordant for CFS, probably related to the disease.
Consequently, we believe the proteomic approach could be useful both to define a panel
of potential diagnostic biomarkers and to shed new light on the comprehension of the
pathogenetic pathways of CFS.

Keywords:

Background

Chronic Fatigue Syndrome (CFS), also termed Myalgic Encephalomyelitis, is a severe,
systemic illness characterized by debilitating, unexplained, persistent fatigue that
indeed has wide impact on the quality of life [1-3]. Besides the disabling fatigue, CFS is accompanied by several symptoms that differ
among patients: fatigue not improved by rest; headaches; sleep disturbances; difficulties
with concentration; memory problems; muscle weakness; musculoskeletal pain, etc. What
is more is that all these symptoms may be aggravated by physical or mental activity
[4,5]. Therefore, CFS is a medically unexplained disorder diagnosed through symptoms reported
by the patient and the exclusion of other medical and psychiatric diseases with the
same symptoms. The estimated worldwide prevalence of CFS is 0.4-2.5%, with a peak
age of onset between 20 and 40 years, and with a female to male ratio of 3:1 [6].

This clinical disorder is considered a multi-factorial disease with unknown etiology
and pathophysiology. Genetic studies have been performed in order to characterize
genes that could be useful as CFS biomarkers [7-10]. In particular, it has been proposed that CFS is associated with immunological and
inflammatory diseases [7,8]. The over-expression of pro-inflammatory proteins, such as interferon-γ (INF-γ),
interleukin-1 (IL-1), and tumor necrosis factor- α (TNF-α), has been reported in CFS
[9]. Besides, up-regulated immune-related genes in patients affected by CFS such as lactotransferrin,
defensin-α1, integrins [8], CMRF35 antigen, IL-8, HD protein [10], and cathepsin C [9] are supportive of the notion that CFS is characterized by immune system activation.
Nevertheless, at present, there are no specific markers of inflammation and/or immune
activation which may be useful in the evaluation of patients with CFS, thus making
the treatment more complicated.

Viral infection has been usually proposed as a causative agent, but despite the efforts
in the elucidation of the role of virus, results are controversial due to a lack of
common standard clinical definition and specific biomarkers of the disease [11-16]. Likewise, vaccines have been depicted by some scholars as playing an important role
in CFS onset [17,18], while on the other hand, others scholars have rejected this possibility, affirming
that the vaccine is safe [19,20].

Whilst in 2011 an International Consensus Panel defined the International Consensus
Criteria that identifies the characteristic patterns of the symptom clusters of CFS
[21], the diagnosis is formulated through the patient’s history and exclusion of other
medical causes and psychiatric problems [5]. Thereby, the availability of biomarkers for CFS could be of great usefulness for
clinical research.

Since genetics is presumed to have a role in the etiology of CFS, one area of investigation
is the twin studies which could be useful in elucidating the role of genetic and environmental
factors in CFS. Nevertheless, a recent study of Byrnes et al.[22] on monozygotic twins discordant for CFS, did not identify a biomarker for CFS in
the transcriptome of peripheral blood leukocytes supposing that positive findings
in prior studies may have resulted from experimental bias. Therefore, we moved to
the study of proteins, and, for the first time, we used a proteomic approach to evaluate
in CFS the global changes of whole saliva (WS).

Recently, the number of publications related to salivary proteome have significantly
increased suggesting human saliva as a biological fluid with an enormous potential
to reflect systemic health conditions. Saliva has many advantages in terms of low
invasiveness, minimum cost, and easy sample collection and processing [23]. Moreover, saliva has a less complex protein composition than serum or plasma reducing
the risk of non specific interactions, and at the same time it represents a useful
diagnostic tool since about 30% of blood proteins are also present in saliva. Therefore,
human saliva proteomics have proven to be a novel approach in the search for protein
biomarkers for detection of diseases [23]. In particular, in the last years, we have obtained encouraging results from the
proteomic analysis of human WS in rheumatic diseases [24-28]. Hence, with the present study, we investigated the proteomics salivary profile in
a couple of monozygotic discordant for CFS. Differences of salivary protein expression
in the CFS patient in respect to the healthy co-twin could be strictly correlated
to the disease itself if twins differ only regarding the presence of CFS.

Methods

Study design

This study adopted a phase approach (Figure 1). The first “discovery” phase was aimed at characterising the salivary proteomic
profile of a CFS subject in comparison to his healthy twin brother. In the second
“validation” phase we performed western blot to assess the ability of these candidate
biomarkers to differentiate the CFS from the healthy subject. Finally, we investigated
the biological function of the significantly changing proteins, applying the Ingenuity
Pathway Analysis (IPA) Knowledge base (Ingenuity® System Inc., Redwood City, CA, USA).
This platform enabled us to visualize the potential interactions between the identified
biomarker signatures in saliva and other molecules of interest, and to identify biological
pathways underlying the CFS disease process.

Figure 1.Study design. This study adopted a phase approach. The first “discovery” phase characterized the
salivary proteomic profile of a CFS. The second “validation” phase assessed the differences
of these candidate biomarkers in CFS in respect to healthy subject. Finally, we investigated
the biological function of the significantly changing proteins, applying the Ingenuity
Pathway Analysis Knowledge base.

Patients

The twins were male, non-smokers, with a body mass index (BMI) of 22.5 (the patient)
and 23.5 (the healthy subject) that has not changed over the years. Both brothers
are graduated, and practice the same work, in particular they have a business company.
They have the same educational background and lifestyle (both are married and have
one son). In 2005, when the twins were 31 years old, they both received for the first
time an attenuated influenza vaccine. The twins did not have a history for autoimmune
disease or other medical conditions. Moreover, there were not any differences in social
and other stressors, not even around the time of exposure to the flu vaccine.

Immediately after this vaccine, the patient, who was previously healthy, developed
asthenia, progressive weight loss, forgetfulness and concentration difficulty, sleeping
disorders, dizziness and fever with indeed determined severe work disability. Physical
examination and oral evaluation were negative in both subjects. Questionnaire scores
and scales are shown in Table 1. The twins completed an extensive questionnaire regarding demographics, family history,
CFS and other health symptoms, social environment, and neuropsychological measures.
The patients were classified based on the 1994 Centers for Disease Control and Prevention
CFS case definition [3].

Whole saliva collection

Salivary samples were collected from the patient with CFS and from his monozygotic
healthy twin brother. Their WS was collected both in 2007 and in 2011 when the twins
were 33, and 37 years old, respectively. This means that WS was collected after two
and six years from the flu vaccine respectively, simultaneously and at the same interval
of time. This study was approved by the local Ethics Committee, and an informed consensus
was obtained for diagnostic or clinical purposes.

WS samples were collected early in the morning (between 8 and 11 a.m) according to
a standard protocol [28]. The twins were asked to refrain from eating and drinking 2 h before saliva collection.
No evidence of oral pathologies or inflammatory processes were observed. The brothers
were asked to gently chew a saliva collector sponge (Surescreen Diagnostics LTD; Derby,
UK) for 2 minutes, and the saliva collected was immediately centrifuged at 17,000
g for 20 minutes at 4°C to yield clear samples. Samples were stored at -80°C. Protein
amounts of resulting supernatants were determined using the Bio-Rad DC-protein assay.
Bovine serum albumin (BSA) was used as a standard.

Two-dimensional electrophoresis

Two hundred μg of proteins, for each sample, were filled up to 400 μl in rehydration
solution. Immobiline Dry-Strips (GE Health Care Europe; Uppsala, Sweden); 18 cm, linear
gradient pH 3–10) were rehydrated overnight in the sample and then transferred to
the Ettan IPGphor Cup Loading Manifold (GE Healthcare) for isoelectrofocusing (IEF).
IEF was performed at 16°C and the proteins were focused for up to 70000Vh. The second
dimension (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis; SDS-PAGE) was
carried out by transferring the proteins to 12.5% polyacrylamide gel, running at 16
mA/gel and 10°C for about 16 h. The gels were stained with Ruthenium II tris (bathophenanthroline
disulfonate) tetrasodium salt (SunaTech Inc.; Suzhou, P. R. China) (RuBP) as described
by Aude-Garcia et al.[29]. Briefly, after the electrophoresis, the gels were fixed in 1% phosphoric acid (v/v)
and 30% ethanol for 1h then were stained overnight with 1 mM RuBP in 1% phosphoric
acid and 30% ethanol. After this time the gels were destained for 5 hours in 1% phosphoric
acid and 30% ethanol and rinsed in water prior to acquisition by “ImageQuant LAS4010”
(GE Health Care). The analysis of images was performed using Progenesis Same Spot
(v4.1, Nonlinear Dynamics; Newcastle Upon Tyne, UK) software. This software generates
2DE analyses which are robust and accurate. Briefly, the gels were aligned to place
all spots in exactly the same location, and afterwards, the spot detection produced
a complete data set since all gels contain the same number of spots, each matched
to its corresponding spot on all gels. After 2DE gel alignment and subsequent spot
detection, the software calculated background corrected abundance, by determining
the lowest intensity value of the image pixels outside the spot’s outlined, and subtracting
it from the intensity value of every pixel inside the spot outline. Theses abundances
were then normalized compared to a reference gel in order to obtain a normalized intensity
value for each spot.

The 2DE experiments were performed in triplicate. The spot volume ratios between the
two different conditions were calculated using the average spot normalized volume
of the three biological replicates (http://www.nonlinear.com/webcite). The software included statistical analysis calculations.

2DE statistical analysis

A comparison between CFS and healthy subject was performed. The significance of the
differences of normalized volume for each spot was calculated by the software Progenesis
Same Spot including the Analysis of variance (ANOVA test). The protein spots with
a ≥2-fold spot quantity change and p<0.05 were cut out from the gel and identified
by Nano-liquid chromatography electrospray ionization tandem mass spectrometry (NanoLC-ESI-MS/MS)
analysis.

NanoLC-ESI-MS/MS Analysis by LTQ-Orbitrap Velos analysis

The gel pieces were destained in 100% ethanol during 2 hours. Subsequently, they were
rehydrated with 100 μl of 50 mM ammonium bicarbonate for 15 min and dehydrated with
100 μl of 50 mM ammonium bicarbonate in 30% acetonitrile (AcN) for 15 min.

The gel pieces were then dried for 30 minutes in a Centrivap vacuum centrifuge (Labconco,
Kansas City, USA). The dried pieces of gel were rehydrated for 45 min at 4°C in 20
μl of trypsin porcine (Sigma-Aldrich; MO, USA) solution (6.25 ng/μl in 50 mM ammonium
bicarbonate) and then incubated at 37°C overnight. Extraction of the peptides was
performed with 20 μl of 1% trifluoroacetic acid (TFA) for 30 min at room temperature
with occasional shaking. The TFA solution containing the proteins was transferred
to a polypropylene tube. A second extraction of the peptides was performed with 20
μl of 0.1% TFA in 50% AcN for 30 min at room temperature with occasional shaking.
The second TFA solution was pooled with the first one. The volume of the pooled extracts
were dried completely and finally resuspended in AcN/formic acid 50%/0.1%.

LC-ESI-MS/MS was performed on a linear trap quadrupole (LTQ) Orbitrap Velos from Thermo
Electron (San Jose, CA, USA) equipped with a NanoAcquity system from Waters. Peptides
were trapped on a home-made 5 μm 200 Å Magic C18 AQ (Michrom) 0.1 × 20 mm pre-column
and separated on a home-made 5 μm 100 Å Magic C18 AQ (Michrom) 0.75 × 150 mm column
with a gravity-pulled emitter. The analytical separation was run for 23 min using
a gradient of H2O/formic acid 99.9%/0.1% (solvent A) and AcN/formic acid 99.9%/0.1% (solvent B). The
gradient was run as follows: 0–5 min 95% A and 5% B, then to 65% A and 35% B at 6
min, and 20% A and 80% B at 7 min at a flow rate of 220 nL/min. For MS survey scans,
the orbitrap (OT) resolution was set to 60000 and the ion population was set to 5
× 105 with an m/z window from 400 to 2000. For protein identification, up to five
precursor ions were selected for collision-induced dissociation (CID). For MS/MS in
the LTQ, the ion population was set to 1 × 10e4 (isolation width of 2 m/z), while
as for MS/MS detection in the OT, it was set to 1 × 10e5 (isolation width of 2 m/z).
The normalized collision energies were set to 35% for CID.

Protein identification

Peak lists were generated from raw orbitrap data using the embedded software from
the instrument vendor (extract_MSN.exe). The monoisotopic masses of the selected precursor
ions were corrected using an in-house written Perl script [30]. The peaklist files were searched against the UniProtKB/Swiss-Prot database (Release-2011_08
of 21-Sep-2011) using Mascot (Matrix Sciences, London, UK). Human taxonomy (20323
sequences) was specified for database searching. The parent ion tolerance was set
to 10 ppm. Variable amino acid modifications were oxidized methionine and fixed amino
acid modifications were carbamidomethyl cysteins. Trypsin was selected as the enzyme,
with one potential missed cleavage, and the normal cleavage mode was used. The mascot
search was validated using Scaffold 3.6.0 (Proteome Software, Portland, OR). Only
proteins matching with two different peptides with a minimum probability score of
95% were considered identified.

Western blot analysis

To validate the expression changes found with 2DE, we used western blot analysis.
Mono-dimensional western blot was performed as previously described [31] for 14-3-3 protein zeta/delta, cyclophilin A (CYPA), Cystatin-C, and Protein S100-A7.
Briefly, aliquots of WS samples were run on 12% SDS-PAGE gels (14-3-3 protein zeta/delta,
CYPA, Cystatin-C) or on 15% SDS-PAGE gels (Protein S100-A7). The amount of proteins
loaded and the dilution of primary antibody was different depending on each protein
analysed (14-3-3 protein zeta/delta and CYPA: 50 μg of proteins and dilution of 1:500,
Cystatin-C 20 μg and a dilution of 1:200, Protein S100-A7 40 μg and 1:200). The immunocomplexes
were detected using a peroxidase-labelled secondary antibody (goat anti-rabbit, 1:10000
dilution; goat anti-mouse, 1:20000; donkey anti-goat, 1:5000). Immunoblots were developed
using the ECL detection system. The chemiluminescent images were acquired by LAS4010
(GE Health Care).

To visualize the different expression of zinc-alpha-2-glycoprotein (ZAG), aliquots
of WS corresponding to 300 μg of proteins, were separated by 2DE using 3–10 linear
strips (18 cm) and 12,5% gels before western blot analysis with specific antibody
(1:1000 dilution).

Western blot statistical analysis

The antigen-specific bands were quantified using the Image Quant-L (GE Health Care)
software. Data were reported as optical density and subjected to statistical analysis
with Student t-test making a comparison of protein expression levels between CFS and
control.

Signaling pathway analysis

Proteins differentially expressed, were functionally analyzed using the Ingenuity
Pathways Analysis (IPA) software v14400082 (2012 Ingenuity Systems, Inc. http://www.ingenuity.comwebcite) with the aim to determine the predominant canonical pathways and interaction network
involved. Swiss-Prot accession numbers and official gene symbols were inserted into
the software along with corresponding comparison ratios between the CFS patient and
the healthy brother. The network proteins associated with biological functions and/or
diseases in the Ingenuity Pathways Knowledge Base were considered for the analysis.
These networks are scored for degree of relevance with values >3 having a 99.9% confidence
level of not being generated by random chance alone. The created genetic networks
describe functional relationships among proteins based on known associations in the
literature.

Results

Proteomic analysis and validation by western blot

Figure 3 illustrates the salivary profiles of the healthy twin brother (A) and of the patient
(B). Overall, we found 13 spots differently expressed in CFS in respect to the control
with a fold variation ≥ 2. Nine spots were up-regulated in CFS and 4 down-regulated.
A list of identified proteins, with molecular weight (MW), isoelectric point (pI),
coverage and score values of NanoLC-ESI-MS/MS is given in Table 3. Table 4 shows the statistical analysis: the normalized volume (mean ± SD) of each spot with
p-value and fold-change in expression levels between samples.

Figure 3.2DE patterns of human saliva. 2DE patterns of human WS: healthy subject (A) and patient with CFS (B). A total of 200 μg of proteins was separated by 2DE using 18 cm pH 3-10L strips and
12.5% SDS-PAGE. The 13 spots differently expressed in CFS in respect to control are
indicated.

Table 3.MS/MS data of salivary proteins found differentially expressed between CFS patient
and his healthy twin

Western blot analysis with specific antibodies was used to validate the expression
changes of four proteins: 14-3-3 protein zeta/delta (n°1051), CYPA (n°1509), Cystatin-C
(n°1575), and Protein S100-A7 (n°1621) (Figure 4A, 4B). The immunoblot for each protein tested is shown in Figure 4 panel C. For each protein the optical density of specific immunoreactive band was
determined and the resulting values compared (Figure 4D). With 2DE we found a significant increase in CFS of 14-3-3 protein zeta/delta,
CYPA, and Protein S100-A7 with a p-value of 0.0009, 0.02, 0.004 respectively (Figure 4B) that was confirmed with western blot. Also the decrease of Cystatin-C (p-value=0.001)
was confirmed by western blot analysis. These experiments of validation were carried
out on WS collected both in 2007 and in 2011, when the twins were 33 and 37, and in
both case the results supported those of the 2DE study.

Figure 4.Proteins differentially expressed in CFS saliva. Enlarged images of 4 proteins found up- or down-regulated in WS of CFS patient in
respect to control subject (A), and histograms of the normalized volume (M±SD) obtained with 2DE analysis for these
proteins (B). Western blot analysis of these 4 proteins in WS collected both in 2007 and in 2011
(C), and the respective histograms of the optical density (OD). Each bar represents the
mean±SD (D). As depicted in Figure 4D, the modulation illustrated in Figure 4B has been confirmed. (* p< 0.05, ** p< 0.01, *** p< 0.001).

Moreover, we conducted mono-dimensional western blot analysis also for ZAG, but the
lower value in the twin with CFS was not significant with a fold variation of 2.0
and 1.2 in WS collected in 2007 and 2011 respectively (Figure 5 panel B). Actually, by 2DE we found two close spots corresponding to ZAG (n°1736,
n°1736A) (Figure 5 panel A, and Table 3), and the mono-dimensional electrophoresis could not allow us to appreciate the real
difference of this protein in WS. Therefore, we performed 2DE immunoblotting to evaluate
the differences of expression observed in different spots. The detected immunoreactive
spots were four and they corresponded to pI values ranging from 5.4 (spot 1) to 5.9
(spot 4) (Figure 5 panel C). The significant decrease in CFS in respect to control was observed for
all spots detected by 2DE immunoblotting confirming the result obtained by 2DE and
NanoLC-ESI-MS/MS.

Figure 5.Expression of ZAG. Enlarged image of ZAG in 2DE gel with the histogram of the normalized volume (M±SD)
(A), and validation of ZAG with mono-dimensional western blot (B), and 2DE western blot (C). With 2DE immunoblotting we found 4 spots corresponding to ZAG (spot 1- spot 4) with
pI values ranging from 5.4 (spot 1) to 5.9 (spot 4). The decrease in CFS in respect
to control was observed for all spots detected by immunoblotting. (* p< 0.05, ** p<
0.01, *** p< 0.001, ns not significant).

Potential Biomarkers found in CFS

Table 5 summarizes the potential biomarkers found in CFS with their functional properties.
Moreover, we performed a preliminary validation on a larger number of patients for
one of these proteins: CYPA (data not shown). ELISA assay for CYPA was carried out
on WS of 20 patients affected by CFS, and 20 healthy subjects, confirming the up-regulation
of this protein: the p-value was 0.03, and the fold variation of this protein in CFS
respect to controls was 2.4.

Table 5.Functional properties of salivary proteins found differentially expressed between
CFS patient and his healthy twin

Pathway and network analysis

The 13 proteins that distinguish the WS of the CFS patient from the healthy subject
were used for bioinformatic analysis to identify biological function and pathways
involved in CFS using IPA. IPA software allows to investigate the relationship between
those proteins and various biological pathways. Figure 6 shows the predominant canonical pathways generated by uploading proteins in Table 4. The software generated different networks. The one with the highest score (score
value 21) encompassed 35 proteins and was associated with “Cellular movement, Hematological
System Development and Function, Immune Cell Trafficking”. Eight of these proteins
were found in our analysis. The top function linked with these biomarkers was the
inflammatory response (p-value 3.78E-05-4.99E-02) that is associated with 8 of our proteins.

Figure 6.Network analysis of differentially expressed proteins. The canonical pathway of differentially expressed proteins with the highest score,
obtained by using IPA. Proteins increased in CFS are colored in red and those that
decrease in abundance relative to healthy control are colored in green.

Discussion

This work, for the first time, used a proteomic approach to evaluate the global changes
in WS in a patient with CFS. In our large cohort of patients with CFS, we selected
a patient with a monozygotic twin brother; despite the two subjects had the same genetic,
education, lifestyle and practiced the same work, one of them developed CFS after
a vaccine. Despite this pilot study analysed only two subjects, the differences of
salivary protein expression could be linked to the disease itself, since the twins
differed only regarding the presence of CFS. By this way, our study on a couple of
twins could permit the focusing on some biomarkers representing the prelude to a targeted
search for these proteins in a wide number of patients, in order to confirm their
usefulness in the diagnosis and therapy of CFS. At present, few proteomics works on
CFS have been carried out on cerebrospinal fluid [32,33], and on serum [34] while in our study we decided to explore the WS of CFS patient preferring to adopt
a less invasive procedure in collecting the samples. Although WS is a less complex
fluid, it has been proved to be a promising diagnostic tool [23-28], and we found an alteration of protein patterns in the patient in respect to his
control.

Immune system

Concerning the proteomic analysis on WS, our results showed an increase of CYPA with
a fold of 2.6, which was also confirmed by the western blot analysis and verified
in WS collected in different years. CYPA was previously found up-regulated in WS of
patients affected by Sjögren’s syndrome and Systemic sclerosis [28,35] sustaining the role of CYPA in the pathogenesis of immune-mediated disorders [36]. Moreover, viral infections have been discovered to promote the secretion of CYPA
[37-39] supporting the hypothesis that a persistent viral infection may contribute to the
pathogenesis of CFS [11].

Besides, the decrease of Ig alpha-1 chain C and of polymeric immunoglobulin receptor
(pIgR) that we observed could have promoted the viral infection/immunological alteration.
In fact, Ig alpha-1 chain C has a role in preventing access of foreign antigens to
the general immunologic system. PIgR ensures humoral defense in the mucosa against
incoming pathogens [40] and it is responsible for intracellular neutralization of some viruses through its
secretory component [41]. For example, Takiguchi and colleagues observed a reduction in pIgR in dextran sodium
sulfate-induced colitis (DSS) [42] supporting the preventive role of pIgR proposed by Murthy et al.[43]. DSS is a model that has been widely used to investigate the mechanisms of inflammatory
bowel diseases, which are chronic inflammatory disorders [42]. The mucosal immune system is primarily responsible for preventing infection caused
by luminal microorganisms by inhibiting bacterial attachment or invasion, and it has
been proposed that pIgR may be necessary for maintaining the basal tone of innate
immunity in the intestinal environment [42,43]. Therefore, these studies support the involvement of pIgR in the protection against
environmental antigens [42,43].

Furthermore, the altered regulation of the immune system in CFS patients is one of
the area of interest in the research of biomarkers for CFS. Brenu and colleagues found
immunological abnormalities, in particular a reduced cytotoxic activity of innate
immune cells, which they proposed as a potential diagnostic tool for CFS [44,45].

In this direction the increase of Human S100A7 (psoriasin) that is overexpressed in
inflammatory diseases could be also seen.

Inflammatory response

Psoriasin has been associated with increased inflammatory cell infiltrates in breast
cancer and various inflammatory disorders [46-49]. As shown by the pathway analysis, the network built with our proteins highlights
the involvement of inflammatory response in CFS pathogenesis.

Among the proteins changed in CFS in respect to the healthy subject, we found two
proteins which belong to the cysteine proteinase inhibitors family: cystatin C and
B. Cystatins function as tight-binding inhibitors of cathepsins, which are cysteine
proteinases involved in a number of important cellular processes including inflammation
[50]. A broad spectrum of biological roles have been suggested for cystatins, including
a role in protein catabolism, in the regulation of hormone processing and bone resorption,
in inflammation, in antigen presentation and T-cell dependent immune response as well
as resistance to various bacterial and viral infections [50]. Salivary cystatins are likely to exert certain antiviral effect as they can interfere
with events in viral replication [47-52]. Our results showed an up-regulation of cystatin B, and a down-regulation of cystatin
C. These two cystatins belong to two different subtypes of cystatin, type I and II
respectively. It was demonstrated that they have different functions, e.g., type I
cystatins are up-regulated in tumor tissue while type II cystatins are generally down-regulated
in tumors [50]. Therefore, we believe our findings could suggest that the balance between cysteine
proteinases and their inhibitors is impaired in CFS.

Signal transduction

In the WS of our CFS patient, we detected the significant increase of two members
of 14-3-3 family in respect to his healthy brother. This family is a class of phosphoserine/phosphothreonine
(pSer/Thr)-recognition proteins implicated in the regulation of many intracellular
signaling [53]. Many organisms express multiple isoforms, and we found isoforms sigma and zeta/delta.
Therefore, 14-3-3 proteins are involved in a wide range of pathological processes
[53] and by means, the increase is probably not CFS-specific. For example, we previously
found an up-regulation of 14-3-3 proteins also in rheumatoid arthritis [26]. In addition, Matsuo et al. have recently found 14-3-3 protein level increased in a patient with Sjögren’s syndrome,
but the patient was originally suspected to suffer from Creutzfeldt-Jakob disease
(CJD) [54] since 14-3-3 protein was considered an important diagnostic marker of CJD [55]. So they supported the assumption that 14-3-3 is non-specifically increased in various
diseases.

Metabolism

Finally, we observed a down-regulation in CFS of ZAG. We found two spots in 2DE gels
corresponding to this protein and, since the specific antibody has a greater sensitivity
in respect to any staining protocol, with western blot we found two other spots, all
confirming the decrease. ZAG is mainly known as an adipokine responsible for lipid
degradation that causes loss of adipose tissue in cancer cachexia. However, beyond
this action, a role of ZAG in the activation of AMP kinase (AMPK), an important regulator
of energy metabolism, in human skeletalmuscle cells has emerged [56]. The mechanism may be involved in mediating the effects of ZAG in relation to increased
energy utilization. This is interesting if we consider that several studies suggest
an organic cause for CFS related to defects in oxidative metabolism. In particular,
individuals with CFS reach exhaustion (in the presence of reduced sarcoplasmic ATP
concentrations) much more rapidly in respect to healthy subjects with resultant acceleration
of glycolysis in working skeletal muscles [57]. Moreover, one of the primary symptoms of CFS is muscle pain and weakness; this process
is probably the result of cellular changes [57] such as reduction in the number of motor units and atrophy due to disuse. Considering
that Russell and colleagues have demonstrated the ability of ZAG to reduce reactive
oxygen species (ROS) production and to counter muscle atrophy associated with insulin
resistance and other catabolic conditions [58], the decrease of ZAG that we found in WS seems to support the hypothetical role of
oxidative stress in CFS. From the same point of view we can explain the increase of
6-phosphogluconate dehydrogenase in WS of CFS. This is an enzyme of the oxidoreductase
class that allows the production of NADPH which is necessary for protection against
ROS.

Conclusions

In conclusion, this study on a couple of monozygotic twins discordant for CFS point
out some proteins which are useful both to define a panel of potential diagnostic
biomarkers and to shed new light on the comprehension of the pathogenetic pathways
of CFS. There is a clear need to extend our results on a larger number of subjects
with similar medical history, and our preliminary results are encouraging for our
future study.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

FC, LG and AL designed the study, coordinated the research, analyzed data and wrote
the manuscript; FC, YDV and ED carried out proteomic analysis; AC and PS carried out
the neurocognitive tests; CG and FS carried out the clinical biochemical analysis;
FM carried out the virological examination; LB carried out the clinical evaluation
of the patient, participated in the design and coordination of study and helped to
draft the manuscript. All authors read and approved the final manuscript.